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目的:研究金黄色葡萄球菌中Hfq蛋白的聚体形式以及聚体和单体的不同生物学功能。方法:利用pET28-(a)载体克隆表达金葡菌来源的Hfq蛋白,通过天然凝胶电泳和SDS-PAGE检测不同条件下Hfq蛋白的存在形式。通过点突变的方法获得56和63位突变的Hfq蛋白,利用凝胶阻滞试验检测单体和聚体的RNA结合活性。结果:Hfq蛋白以单体、二聚体、四聚体以及六聚体的形式存在,各种聚体比例不同,长时间的高温、强酸和强碱会影响聚体的稳定性。在常规SDS-PAGE条件下,重组蛋白仅以四聚体形式存在。突变体蛋白不能够形成聚体且只有形成聚体的Hfq蛋白才能特异结合RNA。结论:Hfq蛋白形成聚体的过程是二聚体四聚体六聚体,且不同聚体形成的机制不同。56位和63位酪氨酸是Hfq蛋白聚体形成的关键氨基酸。聚体结构对Hfq蛋白发挥RNA伴侣分子生物学功能至关重要。
OBJECTIVE: To study the aggregate form of Hfq protein in Staphylococcus aureus and the different biological functions of monomers and monomers. Methods: The Hfq protein of Staphylococcus aureus was cloned and expressed by pET28- (a) vector, and the existence of Hfq protein under different conditions was detected by natural gel electrophoresis and SDS-PAGE. The Hfq proteins with mutation at 56 and 63 were obtained by point mutation. The RNA binding activity of monomers and aggregates was detected by gel retardation assay. Results: The Hfq protein exists in the form of monomer, dimer, tetramer and hexamer. The proportions of various kinds of polymers are different. The prolonged high temperature, strong acid and strong alkali will affect the stability of the polymer. Under conventional SDS-PAGE conditions, the recombinant protein exists only as a tetramer. Mutant proteins are not capable of forming a polymer and only the Hfq protein that forms a polymer binds specifically to RNA. CONCLUSION: The process of Hfq protein forming dimer is dimer tetramer hexamer, and different aggregates form different mechanism. Tyrosine at positions 56 and 63 are key amino acids formed by Hfq protein aggregates. Homopolymerization of Hfq proteins play an important role in the molecular biology of RNA chaperones.